Lipoxygenases are a class of non-heme iron-containing enzymes which regio- and stereospecifically oxidize polyunsaturated fatty acid substrates such as arachidonic acid (AA) and linoleic acid (LA). (Solomon, et al. Chem. Biol. 1997, 4, 795-808; Brash, J. Biol. Chem. 1999, 274, 23679-23682.) The position at which these cis, cis-1,4-pentadiene substrates are oxidized correspond to the requisite lipoxygenase, with the three major human lipoxygenases: 5-LOX, 12-LOX, and 15-LOX-1, oxidizing the C-5, C-12 and C-15 positions respectively. Lipoxygenases are involved in the first committed step in a cascade of metabolic pathways and the products of these enzymes (eicosanoids) are precursors of hormones such as leukotrienes and lipoxins, which mediate a wide array of cellular functions. (Serhan, et al. Chem. Rev. 2011, 111, 5922-5943.) Consequently, the lipoxygenase enzymes and their bioactive metabolites (e.g. hydroxyeicosatetraenoic aicd (HETE) and leukotriene A4) have been implicated in a variety of inflammatory diseases and cancers.
Specifically, 5-LOX has been implicated in cancer and inflammatory diseases, such as asthma and remains the only lipoxygenase enzyme for which there is an FDA-approved inhibitor (Zilueton) on the market.
Reticulocyte 15-LOX-1 has a role in atherogenesis, neurodegenerative diseases, and neuronal damage associated from an acute ischemic stroke event.
12-LOX exists as three isozymes, platelet-type, leukocyte, and epidermal, but leukocyte 12-LOX is found in rat, mouse, pig and cow, but not in humans. (Yamamoto Biochim. Biophys. Acta. 1992, 1128, 117-131; Funk et al. FEBS Lett 1997, 402, 162-166).
Skin Disease and Platelet Hemostatis: Further, 12-LOX has demonstrated a role in skin diseases and platelet hemostasis.
Transplants: 12-LOX inhibitors also have utility in transplantation/xenotransplantation scenarios, where, for example, islets can be treated ex vivo to improve survival prior to transplant.
Cancer: Platelet-type 12-(S)-LOX (12-LOX) has been found to be overexpressed in a variety of tumor tissues including prostate cancer, colorectal cancer, breast cancer and lung cancer. (Catalano et al. Histol. Histopathol. 2005, 20, 969-975; Nie, D et al. Cancer Res. 1998, 58, 4047-4051; Natarajan et al. J. Clin. Endocrinol. Metab. 1997, 82, 1790-1798; Kamitani et al. Adv. Exp. Med. Biol. 1999, 469, 593-598; Soriano et al. Cancer Res. 1999, 59, 6178-6184). Moreover, 12-HETE levels have been linked to increased cancer cell metastasis by facilitating tumor cell motility and angiogenesis. (Nappez et al. Cancer Lett. 1995, 96, 133-140; Timár et al. Int. J. Cancer, 1993, 55, 1003-1010; Honn et al. Exp. Cell. Res. 1994, 214, 120-130; Nie et al. Blood 2000, 95, 2304-2311).
Diabetes: Type 1 and Type 2 diabetes are serious disorders that can lead to major complications and reduced lifespan. There is an unmet medical need in identifying new ways to protect beta cells in these metabolic disorders. 12-LOX is expressed in human pancreatic islets, which is upregulated and activated by inflammatory cytokines leading to increased 12-LOX translocation. The resulting 12-HETE product leads to reduced insulin secretion, reduced metabolic activity and pancreatic β cell death through the amplification of the inflammatory response. (Chen et al. Diabetologia 2005, 48, 486-495). Both non-obese diabetic (NOD) 12-LOX and 12-LOX knock-out (“KO”) mice showed significant resistance to the development of diabetes compared to the controls, showing 12-LOX is a regulator in this disease.
Diabetic Kidney Disease: Further, studies show that activation of the 12-LOX pathway plays a role in the development of diabetic kidney disease (diabetic nephropathy) by multiple pathogenic mechanisms, including decreased expression of glomerular P-cadherin. (Guo, Q. et al. Am. J. Physiol. Endocrinol. Metab. 2011, 300, E708-E716).
Diabetic Nerve Disease: It has also been shown that increased aldose reductase, the first enzyme of the sorbitol pathway, activity plays a key role in diabetes-associated 12/15-LOX activation in the peripheral nerve and spinal cord. (Stavniichuk, R. et al Biochem Pharmacol. 2012, 83, 932-940). Thus, inhibiting 12-LOX is attractive in treating diabetic nerve disease.
Diabetes and Cardiovascular Disease: A selective 12-LOX inhibitor would provide a new therapeutic approach to prevent and/or treat either form of diabetes (type I and type II). The development of 12-hLOX inhibitors provide a potent intracellular approach to decreasing the ability of platelets to form large clots in response to vessel injury or activation of the coagulation pathway. Thus, 12-hLOX inhibition has the ability to attenuate platelet-mediated clot formation caused by diabetes and/or cardiovascular disease and significantly decrease the occurrence of myocardial infarction, congestive heart failure, and stroke. Additionally, studies show that the gene Alox15 that encodes the proteins 12-LOX and 15-LOX are up-regulated in heart failure. Thus, inhibition of 12-LOX could be a treatment for heart failure. (Kayama, Y. et al. J. Exp. Med. 2009, 206, 1565-1574).
Thrombosis: 12-LOX and its product 12-HETE have been implicated in the modulation of hemostasis and thrombosis via their role in regulating platelet function (reactivity, clot formation, calcium mobilization). (Brash, A. R. Circulation 1985, 72, 702-707). Additionally, FcγRIIa is the receptor on the human platelet responsible for heparin induced thrombocytopenia (HIT). It has been found that 12-LOX is essential for FcγRIIa-induced PLCγ2 activity leading to activation of calcium mobilization, Rap 1 and PKC activation, and subsequent activation of the integrin α11bβ3, which demonstrates the role of 12-LOX inhibitors in treating HIT. (Yeung, J. et al. Blood 2014, (DOI 10.1182/blood-2014-05-575878)). Further, 12-LOX has demonstrated a role in skin diseases and platelet hemostasis.
Alzheimer's disease: 12-LOX and 15-LOX are widely expressed in the central nervous system and have been reported to be involved in neurobiology of Alzheimer's disease because it modulates amyloid beta and APP processing. It has also been found that 12-LOX and 15-LOX modulate endogenous tau metabolism, making it an attractive therapeutic for treating Alzheimer's and related diseases. (Giannopoulos, P. F., et al. Aging Cell 2013, 12, 1082-1090).
Non-Alcoholic steatohepatitis: It has been shown that disruption of the gene encoding for 12-LOX, Alox15, protected mice against hepatic steatosis, insulin resistance, and inflammation in experimental liver disease of metabolic origin. (Martinez-Clemente, M. et al. Hepatology 2010, 52, 1980-1991; see also Tanaka, N. et al. Hepatology 2012, 56, 118-129).
One difficulty in being able to clearly define the role of 12-LOX in these systems has been the lack of potent and selective 12-LOX small molecule inhibitors.
A previously reported 12-LOX inhibitor, an 8-Hydroxyquinoline based compound (ML127), exhibited excellent selectivity, >50-100 fold selectivity over related lipoxygenases and cyclooxygenase. In contrast to many of the previously reported inhibitors, kinetic experiments revealed that ML127 was a non-competitive and non-reductive inhibitor. Chiral HPLC separation of the probe molecule revealed a chiral preference for activity with the (−)-enantiomer being much more potent than the (+)-enantiomer (<0.5 μM vs. >25 μM, respectively). (Kenyon, V. et al. J. Med. Chem. 2011, 54, 5485-5497.) However, the chemical series was difficult to optimize further, given that subtle structural modifications led to diminished activity.
There exists a need for a potent, selective 12-LOX small molecule inhibitor that can be optimized without reducing activity to treat or prevent 12-LOX mediated diseases and disorders. The small molecule inhibitor should be soluble, have favorable ADME properties, and have good in vivo PK properties.
Platelets express three immunoreceptor tyrosine-based activation motif (ITAM) containing transmembrane receptors (glycoprotein VI (GPVI)/FcRy complex, C-type lectin-like receptor 2 (CLEC-2), and Low affinity immunoglobulin gamma Fc region receptor II-a (FcγRIIa)). Ligation of ITAM containing receptors on the surface of platelets leads to a shared downstream signaling pathway culminating in platelet activation. These receptors engage in various degrees of hemostasis and thrombosis; however, they have non-redundant (patho) physiological functions. FcγRIIa, a broadly expressed immunorecptor which is present on the surface of human but not mouse platelets is best known for its pathophysiological role in immune-mediated thrombocytopenia and thrombosis; a family of disorders including immune thrombocytopenia, thrombocytopenia associated with sepsis, and heparin-induced thrombocytopenia (HIT). Selectively inhibiting the FcγRIIa signaling pathway in platelets for prevention of immune-mediated thrombocytopenia and thrombosis has been a long sought approach for prevention of HIT (Reilly et al., Blood 2011, 117, 2241-2246).
Heparin is widely used in the clinic to treat thrombosis. However, more than 3-5% of patients taking heparin will develop an immune response to heparin and be at high risk for heparin-induced thrombocytopenia (HIT), which can lead to a life-threatening thrombotic event mediated by the immune system. The current therapeutic approach to treatment of HIT is the removal of heparin treatment and replacement with direct thrombin inhibitors (DTIs) which have an inherently high risk for serious bleeding and must be monitored in the clinic. Even with this potentially fatal complication, heparin remains the standard anticoagulant for prevention and treatment of thrombosis.
Therefore, there is also a need for novel therapeutic approaches that directly target the pathogenesis of HIT.